Research ArticleStudy on the Multitarget Synergistic Effects of Kai-Xin-San againstAlzheimer’s Disease Based on Systems Biology

Sirui Guo,1,2 Jiahong Wang ,3 Yunjia Wang,1 Ying Zhang,1 Kaishun Bi,1 Zhou Zhang ,3

and Qing Li 1

1School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, 110016 Liaoning, China2Department of Pharmacy, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy ofMedical Sciences, Beijing 100730, China3School of Life Sciences and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, 110016 Liaoning, China

Correspondence should be addressed to Zhou Zhang; zzhouzhang@163.com and Qing Li; lqyxm@hotmail.com

Received 29 August 2019; Revised 10 November 2019; Accepted 29 November 2019; Published 31 December 2019

Academic Editor: Luciana Mosca

Copyright © 2019 Sirui Guo et al. This is an open access article distributed under the Creative Commons Attribution License, whichpermits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Kai-Xin-San (KXS), a classical Chinese traditional prescription, was widely applied in the treatment of Alzheimer’s disease (AD),while its functional mechanisms still remain unclear. By using systems biology approaches at animal, cellular, and molecular levels,the improvement of KXS on cognitive impairment was achieved by inhibiting abnormal acetylcholinesterase. The function on thenerve skeleton was performed by regulating the Tau phosphorylation pathway. Its antioxidant, anti-inflammatory, andantiapoptotic effects by modulating the aberrant upregulation of ROS, proinflammatory factors, and apoptosis-related proteinsin the brain were studied to reveal the synergistic therapeutic efficacy of KXS. Then, formula dismantling in vitro indicated thatginseng was the principal herb, whereas three other herbs served adjuvant roles to achieve the best effect. After that, the in vivoanalysis of components into plasma and brain of AD rats showed that 8 of 23 components in blood and 4 of 10 components inbrain were from ginseng, respectively, further verifying the principal status of ginseng and the synergistic effects of the formula.Thus, the anti-AD effects of KXS were achieved by multitargets and multichannels. The systems biology approaches presentedhere provide a novel way in traditional herbal medicine research.

1. Introduction

Herbal medicine formulae with unique advantages have beenwidely used for thousands of years based on the guidance oftraditional Chinese medicine (TCM) theory, a combinedtherapeutic system in fighting disease [1]. Generally, a for-mula is composed of several Chinese herbal medicines. Theherbs with the strongest pharmacological action have a prin-cipal role, while others play an assisting role to strengthen theeffect or minimize the adverse effects [2]. It is well knownthat Chinese herbal formulae have holistic treatment andsynergy strategies; there are many components in TCM [3].However, how these herbs can be combined in one formulaand create a powerful therapeutic effect still remains confus-ing. Therefore, it is important to study the synergistic effects

and the underlying mechanisms of Chinese herbal medicineformulae.

Alzheimer’s disease (AD), a widespread neurodegenera-tive disease, is characterized by the deposition of intercellularamyloid polypeptides and intracellular neurofibrillary tangles(NFTs), which are mainly composed of hyperphosphorylatedTau [4]. AD is also triggered by other complicated pathoge-netic factors such as oxidative stress, neuroinflammation,and neuronal apoptosis, and there is a vicious circle amongthem inmany patients [5]. It was reported that there are morethan 50 million cases of dementia worldwide in 2018, signify-ing that one new case occurs every 3 seconds [6]. The increas-ing prevalence of dementia not only caused pain to thepatients but also imposed a heavy burden on the affected fam-ilies and the whole society [7, 8]. The annual total cost of

HindawiOxidative Medicine and Cellular LongevityVolume 2019, Article ID 1707218, 15 pageshttps://doi.org/10.1155/2019/1707218

dementia was $167.74 billion in 2015, and it is predicted toreach $507.49 billion in 2030 and $1.89 trillion in 2050[9, 10]. Currently, all FDA-approved drugs were developedfor a specific single target, such as cholinesterase inhibitors(donepezil, rivastigmine, and galantamine) and N-methyl-D-aspartate receptor agonist (memantine) [11]. However,their therapeutic effects were below our expectationsbecause their actions on a single target could not matchmultiple pathogenetic factors mainly including abnormalβ-amyloid and Tau [12–14]. Interestingly, with the multi-target and multicomponent treatment characteristics, TCMhad unique advantages in dealing with AD [15, 16]. It hasbeen proven that Panax ginseng, one of the main representa-tives of TCM, was effective in treating AD, and the key mech-anism has been revealed to be related to the PI3K/Aktsignaling pathway [17]. A series of formula studies on thePanax ginseng/Polygala tenuifolia combination yielded vary-ing improvements in the course of AD and the most repre-sentative formula was Kai-Xin-San (KXS), initially recordedin Bei Ji Qian Jin Yao Fang in Tang dynasty (AD 652)[18, 19]. KXS consists of four single herbs which are usedin treating AD frequently, namely, Polygala tenuifolia Willd.(PR), Panax ginseng C. A. Mey. (GR), Poria cocos (Schw.)Wolf (PO), and Acorus tatarinowii Schott (AT) [20]. Recentresearches of KXS were mainly focused on component iden-tification or mechanism exploration, and most pathologicalstudies were related to single pathway neurotransmitter reg-ulation, which could not completely explain the therapeuticmechanism of KXS and the synergistic effects among the foursingle herbs [21, 22]. Therefore, there is an urgent need toexplain the multidimensional beneficial effects and syner-gism of KXS with systems biology methods.

In order to explore the principle of compatibility in TCM,the treatment of AD with KXS was applied as the workingmodel. Aβ25-35- and D-gal-induced rats and Aβ25-35-inducedPC12 cells were applied to establish AD models [23, 24].Hence, we explained the therapeutic effects of KXS at themolecular, cellular, and animal levels and expounded thebiological mechanism and synergism in fighting AD multidi-mensionally, which could provide research strategies instudying traditional formula.

2. Materials and Methods

2.1. Preparation of KXS. The decoction pieces of GR, PR, PO,and AT were purchased from Tongrentang Pharmacy. KXSwas prepared using GR, PR, PO, and AT at a weight ratioof 3 : 2 : 3 : 2, then extracted by double-distilled water(1 : 10 w/v) for 1 h, and repeated a second time. The mixedextracted solutions were concentrated to 1mg/mL by a rotaryevaporator. The extraction method of four single herbs wasconsistent with the formula, and the concentrations werecalculated based on the total dose.

2.2. Experimental Animals. Male Sprague-Dawley (SD) ratsweighing 200-250 g (about 6 weeks old) were obtained fromthe Experimental Animal Center of Shenyang Pharmaceuti-cal University. SD rats were kept in a controlled environment(temperature: 23 ± 2°C; humidity: 50 ± 10%) under a 12 h

light/12 h dark cycle with water and standard diet ad libitum.They were kept for 7 days to adapt to the new surroundings.Rats were randomly divided into eight groups (n = 12).Briefly, the AD rats were injected intraperitoneally withD-gal (50mg/kg/mL) for 6 weeks and the control groupwas given the same volume of saline. Six groups of AD ratswere administered intragastrically with KXS (10.0 g/kg), GR(3.0 g/kg), PR (2.0 g/kg), PO (3.0 g/kg), AT (2.0 g/kg), andHup A (0.027mg/kg) once a day from the third week untilthe end of the experiment [25]. At the end of the fourth week,the stereotaxic injection of 4μg Aβ25-35 (1μg/μL) wasoperated at the following coordinates: anteroposterior—3.5;mediolateral—+2.0; and dorsoventral—2.8mm [26]. Twoweeks later, the Morris water maze test was used for evalua-tion. All rats were sacrificed with intraperitoneal injectionof chloral hydrate (350mg/kg). The experimental operationswere approved by the Guidelines for Animal Experimenta-tion of the Shenyang Pharmaceutical University and theAnimal Ethics Committee of the institution.

2.3. Morris Water Maze Test. At the seventh week, Morriswater maze performance was applied to evaluate a rat’s spa-tial learning and memory ability [27]. The rats were trainedto find a hidden safety platform located in the same position(1 cm below the water surface, 10 cm in diameter), and com-plete training was conducted twice per day for 4 days. Thetraining trial was finished if the rat stood on the safety plat-form and stayed for at least 10 s. Those rats that could notfind the hidden platform within 90 s were guided to the plat-form and kept on the platform for 10 s. The probe test pro-ceeded 24 h after the last training trial. The rats were testedto swim freely to find the removed platform within 90 s.The distance and time in the target quadrant as well as thetimes of crossing the platform were recorded to evaluate spa-tial memory. The drug administration and Morris watermaze test were scheduled according to the experimentaltimelines (Figure 1) and followed the methods of Guo et al.2019 [28].

2.4. Immunofluorescence and Immunohistochemistry. Theexpression of neuronal nuclei (NeuN) was determined withimmunofluorescence. Brain tissue sections (4μm thickness)were rehydrated with gradient alcohol, antigen repairedwith citric acid buffer (pH6.0), and then blocked withBSA for 30min. The sections were incubated with NeuNantibody (Servicebio, 1 : 300) at 4°C overnight, followedby CY3-labeled secondary antibodies (Servicebio, 1 : 500) atroom temperature for 60min. For nucleus staining, the sec-tions were incubated with DAPI fluorescent dye for 8minand mounted with antifade mounting medium. Images ofthe immunofluorescence slices were scanned with NikonEclipse C1. The positive expression in the hippocampal CA1region (3 sections per rat) was dealt with the Image-Pro PlusSoftware.

The expressions of phosphorylated Tau in hippocampusCA1 were operated with immunohistochemical analysis.Histologic sections (4μm thickness) were antigen repairedwith citric acid buffer (pH6.0), incubated with 3% H2O2 for25min, and then blocked with 3% BSA for 30min.

2 Oxidative Medicine and Cellular Longevity

Subsequently, the sections were incubated with monoclonalanti-Tau (phosphor S396) antibody (Abcam, 1 : 1000) at4°C overnight, followed by streptavidin-horseradish peroxi-dase (Servicebio, G1211) for 60min at room temperature.DAB color-substrate solution (Servicebio, G1211) wasapplied for coloring, and the images of the immunohisto-chemical sections were acquired by a CIC microscope(XSP-C204).

2.5. Western Blotting. The hippocampus was harvested andlysed with lysis buffer (Beyotime Biotechnology, China)containing 1% phosphatase inhibitors and protease inhibi-tor. The quantitative proteins (10μg) were separated on10% SDS-polyacrylamide gels and transferred to a PVDFmembrane. The PVDF membrane was blocked with 5%skim milk powder in Tris-buffered saline containing 0.1%Tween 20 (TBS-T) for 2 h and then incubated in the primaryantibodies diluted in blocking solution overnight at 4°C. Thedetails of the primary antibodies are listed in SupplementaryTable 1. Then, the PVDF membrane was washed withTBS-T and incubated in corresponding secondaryantibodies at room temperature for 2h. After that, the PVDFmembrane was covered with ECL and photographed withthe Chemiluminescent Imaging System (Tanon Science &Technology Co., China). β-Actin was calculated as a controlfor protein expression in every membrane.

2.6. Detection of Acetylcholinesterase (AChE) Activity,Proinflammatory Factors, and Reactive Oxygen Species(ROS) In Vivo. Brain tissue sections in the hippocampuswere homogenized with 10 volumes (1 : 10, w/v) 0.1mMphosphate-buffered solution (PBS) and centrifuged at1,000 g for 10min at 4°C. The supernatants were used formeasuring the activity of acetylcholinesterase according tocomplimentary instructions (Nanjing Jiancheng, China).The absorbance was evaluated with a microplate reader at412nm. The AChE activity was calculated as follows: activityðU/mgprotÞ = ½ODsample − ODcontrol�/½ODstandard −ODblank�∗ standard concentration/protein concentrationsample. Thesupernatants were collected to also measure theproinflammatory factor (TNF-α and IL-1β) levels in thehippocampus using an enzyme-linked immunosorbent assay(Elisa) kit (Boster Biological Technology Co. Ltd., China)according to the manufacturer’s detailed instructions. Theabsorbance was evaluated with a microplate reader (TECAN,

Switzerland) at 450nm. The level of ROS was measuredwith the ROS assay kit (Nanjing Jiancheng, China). Accord-ing to the description of a previous study [29], the fluores-cence in fresh hippocampal tissue was evaluated with theexcitation at 500nm and the emission at 525nm.

2.7. Cell Culture, Drug Treatment, Cell Viability,Proinflammatory Factors, and ROS In Vitro. PC12 cells wereobtained from the Boster Biological Technology Co. Ltd.(Wuhan, China) and cultured in H-DMEM supplementedwith 1% L-glutamine, 1% penicillin/streptomycin, and 10%FBS (Gibco). Cells were incubated at 37°C in moist air con-taining 5% CO2. In brief, PC12 cells were seeded in cultureplates (1 × 105 cells/mL) for 24 h. Then, the cells were treatedwith KXS (1mg/mL), GR (0.3mg/mL), PR (0.2mg/mL), PO(0.3mg/mL), and AT (0.2mg/mL), respectively, for 24 h.Subsequently, Aβ25-35 was added to the medium and coincu-bated for an additional 24 h. Cell viability was measured bythe 3-[4,5-dimethylthiazol 2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assay. After different drug treatments, thetreated PC12 was incubated with 100μL MTT (1mg/mL,Sigma-Aldrich) in a culture solution for 4 h and followed bythe addition of 150μL dimethyl sulfoxide (DMSO). Theabsorbance was detected at 490 nm wavelength by a micro-plate reader. Meanwhile, the supernatant after different drugtreatments was collected to determine the level of IL-1β andTNF-α. The concentrations of IL-1β and TNF-α were mea-sured according to the standard protocol from the manufac-turer of Elisa assays (Boster Biological Technology Co. Ltd.,China). The absorbance was evaluated with the microplatereader at 450 nm. Intracellular ROS was measured with theROS assay kit (Beyotime Biotechnology, China). 2,7-Dichlor-ofuorescin diacetate (DCFH-DA) was a sensitive intracellularreactive oxygen detection probe widely used. Then, treatedPC12 cells were loaded with 5μmol/L DCFH-DA for30min at 37°C and washed with PBS for three times. Accord-ing to a previous study [30], the fluorescence in PC12 cellswas evaluated with the excitation at 500nm and the emissionat 525nm.

2.8. Tubulin-Tracker Red Analysis. Aβ25-35-induced cytoskel-etal change of the PC12 cell was evaluated by a laser scanningconfocal microscope. In brief, cells were prepared on 20mmdiameter coverslips in 24-well cell culture plates at 1 × 104cells/well. After the drug treatment, the cytoskeletalchanges of the PC12 cell’s nucleus and microtubules were

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evaluated by fluorescent visualization with DAPI (a bluefluorescent dye) and Tubulin-Tracker Red (a red fluores-cent probe for microtubules), respectively. After the drugtreatment, cells were washed twice in PBS and fixed with4% paraformaldehyde for 20min. Subsequently, the fixedcells were permeabilized twice with 0.1% (v/v) Triton X-100in PBS for 5min, incubated with Tubulin-Tracker Red(1 : 100) for 1 h, and then washed three times in 0.1% (v/v)Triton X-100. For nucleus staining, the fixed cells were incu-bated with DAPI fluorescent dye for 8min and then washedand dried before use. The fluorescently labeled cells wereexamined by a C2 plus laser scanning confocal microscope(Nikon, Japan).

2.9. Measurement of the Characteristic Constituents of KXS InVivo. The plasma samples collected from six rats after theMorris water maze test were mixed to eliminate individualvariation. Aliquots of 1mL mixed plasma samples wereextracted with 3mL methanol by vortex mixing for 3min.After centrifugation at 12000 rpm and 4°C for 5min, thesupernatants were transferred and evaporated to dryness at35°C. Then, the residue was reconstituted in 100μLmethanolfor HPLC-TOF-MS/MS analysis.

The HPLC system (Agilent 1260) and quadrupoletime-of-flight mass spectrometry constituted the HPLC-QTOF-MS system. Chromatographic separation wasachieved with a Phenomenex Kinetex XB-C18 Column(100mm× 4:6mm, 2.6μm) at 30°C. Mobile phases were thewater (a) and methanol (b). A mobile phase was achievedwith the following gradient elutions: 95%A⟶ 75%A at0–10.00min; 75%A⟶ 60%A at 10.00–25.00min; 60%A⟶ 25%A at 25.00–35.00min; 25%A⟶ 10%A at 35.00–41.00min; 10%A⟶ 10%A at 41.00–45.00min; 10%A⟶ 95%A at 45.00–50.00min; and 95%A⟶ 95%A at50.00–55.00min at a flow rate of 0.5mL/min with a sampleinjection volume of 5μL. Mass spectrometric detection wasoperated by a Triple TOF 5600 (AB SCIEX, Foster City,CA) with both negative and positive ESI. The optimizedparameters are listed as follows: ion spray voltage—5500V(positive mode) and -4500V (negative mode); source tem-perature—550°C; nebulizer gas as Gas 1—50psi; heater gasas Gas 2—50psi; curtain gas as Gas 3—30psi; and declus-tering potential—80V (positive mode)/-80V (negativemode). Information-dependent acquisition was operatedwith the mass range of m/z 100–2000 to carry the MS/MSexperiment. The collision energy of 60 ± 15V with a colli-sion gas of 15psi was applied for information-dependentacquisition. The acquisition, instrument control, and analy-sis of data were dealt with Analyst TF 1.6 Software(AB SCIEX, USA).

2.10. Statistical Analysis. All statistical data were analyzed byStudent’s t-test and one-way ANOVA with post hocDuncan’s multiple range test (SPSS 19.0 software). TheHPLC-QTOF-MS data acquisition was administered by theAnalyst TF 1.6 Software (AB SCIEX, USA), and the data werefurther processed with the PeakView Software (version 2.2,AB SCIEX, USA). The statistical results were expressed

as mean ± SD, and statistical significances were classifiedas P < 0:05.

3. Result

3.1. Therapeutic Efficacies of KXS on Cognition Impairment ofan AD Rat Model. In SD rat injected with D-gal and Aβ25-35,a significant decrease of escape latency and swimming dis-tance was observed in rats treated with KXS compared withother groups treated with vehicle control, positive control,or monotherapy of GR, PR, PO, and AT (Figure 2(a)). Inthe single-herb treatments, GR showed the most effectivetherapy. In the spatial probe, the rats treated with KXSshowed a significant increase on the time and swimming dis-tance in the target quadrant to find the removal platform(Figures 2(b) and 2(c)). In addition, KXS group rats showedmore platform crossings than the other treatment groups(Figure 2(d)). Consistent with this, the expression of NeuNin the hippocampal CA1 regions of rats treated withKXS was higher than that in other treatment groups(Figures 2(e) and 2(f)). GR or PR alone also induced theexpression of NeuN to some extent. Like Hup A, the AChEinhibitor, KXS could reduce the activity of AChE. Interest-ingly, GR or PR inhibited D-gal- and Aβ25-35-triggeredaugmentation of AChE, whereas KXS further intensified thiseffect (Figure 2(g)). Previous studies showed that the concen-tration of ACh was significantly reduced in the AD modelwith HPLC-MS/MS quantitative analysis [31]. It was sug-gested that KXS could ameliorate ACh loss (correlated withcognition impairment of AD) by suppressing the activity ofAChE. Furthermore, the KXS protocol did not cause weightloss in AD rats (Figure 2(h)). These results suggested theKXS ameliorate cognition impairment without side effectson the AD rat model.

3.2. KXS Inhibited Oxidative Stress and Neuroinflammation-Induced Apoptosis In Vivo. By fluorescence analysis of theDCF fluorescence intensity in the hippocampus of ratsinduced by D-gal and Aβ25-35, it was found that GR, butnot PR, PO, or AT, induced an apparent decline of ROSlevels, whereas KXS played the most important role com-pared with mono-GR, PG, PO, and AT (Figure 3(a)). It wastested if KXS treatment could act on proinflammatory factorexpression by Elisa detection. Alone, GR had the greatestreduction in the concentration of TNF-α and IL-1β in fourmonoherbs of KXS, whereas KXS further enhanced the anti-neuroinflammation effect (Figures 3(b) and 3(c)). Previousstudies demonstrated that the accumulation of ROS and pro-inflammatory factors triggers the caspase cascade effect andcauses apoptosis in the AD pathological process [32]. Bax,bcl-2, and cleaved-caspase-3 proteins were critical for themitochondria apoptosis pathway and were abnormallyexpressed in AD. It was found that in the hippocampus ofAD rats treated with KXS, bcl-2 at the protein level wasdownregulated, whereas Bax and cleaved-caspase-3 wereupregulated (Figure 3(d)). Further analysis showed that asingle GR or PR ameliorated the abnormal expression ofthese proteins, and the KXS formula enhanced the regulatingeffect. These results suggested that the KXS formula could

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inhibit oxidative stress and neuroinflammation-induced apo-ptosis; GR played the primary role in the formula, whereasPR, PO, and AT acted as adjuvant ingredients.

3.3. KXS Inhibited the Phosphorylation of Tau via PI3K/AktSignaling Pathway In Vivo. Abnormal or hyperphosphory-lated Tau was the principal pathogenetic factor in the patho-genesis of AD [33]. Previous studies showed that GSK-3βwas the major protein kinase to trigger the hyperphosphory-lated Tau in the AD brain [34]. By western blot analysis of theproteins related to the Tau signaling pathway in the ADrat hippocampus, it was found that KXS could upregulatethe expressions of p-PI3K, p-Akt, and p-GSK-3β (S9), fur-ther inhibiting Tau hyperphosphorylation (Figures 4(a)and 4(b)). Among the four single herbs of KXS, GR playedthe principal role in the suppressive effect of hyperpho-sphorylated Tau (S199 and S396). Immunohistochemicalstaining with the anti-p-Tau (S396) antibody was appliedto evaluate the expression of p-Tau in hippocampal CA1regions. It was found that GR and PR could significantlydownregulate p-Tau (S396), whereas the KXS formula fur-ther strengthened this effect (Figures 4(c) and 4(d)). Theseresults suggested that KXS could inhibit the phosphoryla-tion of Tau via the PI3K/Akt signaling pathway in AD ratsand indicate that there could be a synergistic effect in thefour single herbs of KXS.

3.4. KXS Causes Synergistic Effects on Aβ25-35-Induced PC12Cells In Vitro. To further validate the rationality of the foursingle herbs, in vitro researches were performed to test ifPR and/or PO and/or AT combined with the administeringof the GR herb exerted additive or synergic effects onPC12 cells. In PC12 (1 × 105 cells/mL) co-incubated with20μmol/L Aβ25-35, a statistically significant increase of activ-ity was detected in cells treated with the formula KXS com-pared with PC12 cultured with model control or combinedtherapy of GR, PR, PO, and AT. Intriguingly, PR, PO, orAT improved GR-enhanced activity of Aβ25-35-inducedPC12 cells, whereas the KXS formula further promoted thiseffect (Figure 5(a)). Consistent with this, released proinflam-matory factors (TNF-α and IL-1β) in KXS-treated PC12 cellswere lower than in other treatment groups (Figures 5(b)and 5(c)). Cells, upon KXS treatment, showed higher activityto inhibit oxidative stress detected by the ROS levels, andshowed downregulated ROS levels compared with modelcontrol or combined therapy of GR, PR, PO, and AT(Figure 5(d)). Previous studies showed that Tau hyperpho-sphorylation could reduce microtubule stability and influ-ence the expression of Tubulin in the AD model [35]. Itwas found that GR alone ameliorated a certain degree ofthe microtubule morphology. What’s more, the stabilizationeffect on microtubules was further enhanced in Aβ25-35-induced PC12 cells with the increase of compatible herbs

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Figure 2: The ameliorative effect of KXS on AD rat memory and learning impairment. KXS significantly shortened the escape latency andswimming distances (a) of rats bearing cognition impairment compared with those treated with a monotreatment of GR, PR, PO, and AT.KXS prolonged the time (b), swimming distance (c), and platform crossings (d) to find the removal platform in the target quadrant.Treatment with KXS resulted in the high expression of NeuN (e and f) and the activity of AChE (g) in the hippocampus. KXS did notcause loss of body weight, suggesting that KXS might probably not cause severe toxicity (h). The column diagrams marked by differentletters were significant to each other at the 0.05 level according to Duncan’s multiple range test. Values were mean ± SE (n = 6),scale bar = 20μm.

6 Oxidative Medicine and Cellular Longevity

(Figures 5(e) and 5(f)). Consistent with in vivo results, theseresults suggested the synergic effects in the four herbs, andGR played the principal role in the formula, whereas PR,PO, and AT could serve as adjuvant herbs.

3.5. Analysis of the Basic Pharmacodynamic Material of KXSat the Small Molecular Level. After the optimization ofchromatographic separation conditions and MS detectionparameters, an appropriate HPLC-QTOF-MS method wasdeveloped to explore the ingredients of KXS in the AD ratmodel. The multiple product ion filtering (mPIF) techniquebased on multiple diagnostic product ions and the neutralloss filtering (NLF) technique was applied to identify com-pounds in vivo [21]. In the full-scan mass spectra, the com-pounds exhibited [M+H]+ or [M+Na]+ ions in positive ionmode and [M–H]– in negative ion mode. In total, 23 compo-nents were firstly found in AD rats’ plasma (including 8 fromGR, 1 from PR, 4 from PO, 6 fromAT, and 4 that only existedin KXS) by analyzing blank and drug-containing plasmasamples of AD rats. 10 constituents were firstly detected inAD rats’ brain including 4 from GR and 6 only from KXS.The order of the isomers was preliminarily determinedaccording to polarity. The compounds and their MS dataare listed in Table 1 and Table 2. The peak area of compo-nents in vivo after oral administration of KXS in AD rats ispresented in Supplementary Tables 1–2. The main effectivecomposition in vivo was preliminarily elucidated, and themass accuracy of all identified compounds was less than

5ppm. Based on these, it was indicated that there weresynergic effects among the four single herbs and KXSfacilitated the transportation of GR into plasma and brain.

3.6. Correlation Analysis of Plasma Components andPharmacological Effects of KXS. To explore the relationshipbetween plasma components and pharmacological effects ofKXS, the Pearson correlation analysis was performed withSPSS 19.0 software to rank four single herbs in an anti-ADeffect [36, 37]. To evaluate the absolute value of thecoefficient as the degree of correlation, we performed a semi-quantitative analysis of each prescription chromatogram(Table 3). When the Pearson correlation coefficients weregreater than 0.5 and less than or equal to 0.8, it revealed a sig-nificant correlation; moreover, when it was greater than 0.8,it revealed a high correlation. To intuitively evaluate the con-tribution of four single herbs, 9 plasma components weresuccessfully screened from the detected components basedon the values of the Pearson correlation coefficient(∣r∣> 0.5) as shown in Table 4. The results also providedstrong evidence that GR was the principal herb in the for-mula, whereas PR, PO, and AT served as adjuvant herbs.

4. Discussion

Traditional Chinese medicines (TCM) are frequently used inthe treatment of diseases, which reflects the characteristics ofmulticomponents and multitargets of TCM. At the same

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hipp

ocam

pus

(d)

Figure 3: Effect of KXS on oxidative stress and neuroinflammation-induced apoptosis. KXS treatment relieved the accumulation of ROS inthe rat hippocampus (a). Effect of KXS on representative proinflammatory factors TNF-α (b) and IL-1β (c). Downregulation of apoptosis-related proteins Bax and bcl-2 by KXS treatment in the rat hippocampus resulted in a decrease of cleaved-caspase-3. Values were mean ±SE (n = 6). The column diagrams marked by different letters were significant to each other at the 0.05 level according to Duncan’smultiple range test.

7Oxidative Medicine and Cellular Longevity

(S199)Tau

Tau(S396)

Tau

𝛽-Actin

p-PI3K

PI3K

p-Akt

Akt

GSK-3𝛽

GSK-3𝛽(S9)

Con Model KXS GR PR PO AT Hup A

(a)

0

1

2

3

C C

CC

A

BB B B

BBB B

AB

AB

A

ABAB AB AB

AB

A

aAE DECDCD

ABC

DD

D DDBC

BCAB

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Rel.e

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ssio

n hi

ppoc

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s

p-PI

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kt

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(S9)

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u

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u

ConModelKXSGR

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(b)

(c)

A AB

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E

Con

Mod

el

KXS

GR PR PO AT

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A

ConModelKXSGR

PRPOATHup A

0

5

10

15

20

Rel.

p-Ta

u(S3

96) l

evel

(d)

Figure 4: KXS inhibited the phosphorylation of Tau via the PI3K/Akt signaling pathway in vivo. Downregulation of p-PI3K, p-Akt, andp-Tau and upregulation of p-GSK-3β were shown in the rat hippocampus treated with KXS compared with those treated with amonotreatment of GR, PR, PO, and AT. The efficiency of KXS was detected (a) and quantitated (b) by western blot analysis. Theexpression of p-Tau (S396) in the rat hippocampus was performed with immunohistochemistry analysis (c and d). Each of the controlgroups were adjusted to the same level. The column diagrams marked by different letters were significant to each other at the 0.05 levelaccording to Duncan’s multiple range test. Values were mean ± SE (n = 6), scale bar = 50μm.

8 Oxidative Medicine and Cellular Longevity

0

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(pg/

g)

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A𝛽 (20 𝜇mol/L)

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Figure 5: Continued.

9Oxidative Medicine and Cellular Longevity

Mer

geTu

bulin

DA

PI

Con A𝛽

20 𝜇m 20 𝜇m 20 𝜇m 20 𝜇m 20 𝜇m 20 𝜇m 20 𝜇m 20 𝜇m 20 𝜇m 20 𝜇m

A𝛽

KXB GR+PR+AT GR+PR+PO GR+PR+AT GR+PR GR+AT GR+PO GR

(f)

Figure 5: In vitro therapeutic efficiency of KXS on Aβ25-35-induced PC12 cells. KXS significantly promoted the cell viability of PC12compared with those treated with PR and/or PO and/or AT combined with the administering of the GR herb (a). Treatment with KXSresulted in the inhibition of inflammation revealed by an alleviation of TNF-α (b) and IL-1β (c). Detection of ROS reduction in PC12 cellstreated with KXS (d). Morphological examination of Tubulin in PC12 cells treated with KXS and PR and/or PO and/or AT combinedwith the administering of the GR herb (e and f). The column diagrams marked by different letters were significant to each other at the0.05 level according to Duncan’s multiple range test. Values were mean ± SE (n = 6), scale bar = 20 μm.

Table 1: Identification of the components of Kai-Xin-San in plasma samples of rats.

No. Formula tR (min) Ion addition Source Cal m/z m/z Fragment ions (m/z)

1 C10H12O5 3.60 M+Na½ �+ KXS, AT 235.0577 235.0579 —

2 C12H14O5 31.14 M+Na½ �+ KXS, AT 261.0733 261.0729 —

3 C12H16O4 2.14 M+Na½ �+ KXS, PR 247.0941 247.0949 —

4 C12H16O4 31.30 M+H½ �+ KXS, AT 225.1121 225.1125 —

5 C12H16O5 24.85 M+H½ �+ KXS, PO 241.1071 241.1053 186.9109

6 C12H16O5 40.45 M+H½ �+ KXS, AT 241.1071 241.1071 —

7 C16H20O9 38.04 M −H½ �− KXS, GR 355.1035 355.1016 —

8 C21H24O11 40.12 M+H½ �+ KXS, PO 453.1391 453.1385 —

9 C22H30O14 48.39 M+Na½ �+ KXS, AT 541.1528 541.1535 —

10 C36H46O7 45.95 M+H½ �+ KXS, PO 591.3316 591.3316 —

11 C36H56O13 49.49 M −H½ �− KXS, AT 695.3648 695.3621 —

12 C36H60O8 46.54 M −H½ �− KXS 619.4215 619.4288 —

13 C36H62O10 46.79 M −H½ �− KXS, GR 653.4270 653.4260 277.1787‐257.1526‐231.1734

14 C41H70O13 41.75 M −H½ �− KXS 769.4744 769.4705 339.1989

15 C41H70O14 46.33 M+H½ �+ KXS, GR 787.4838 787.4866 —

16 C42H72O14 45.98 M+Na½ �+ KXS, PO 823.4819 823.4792 662.2742‐479.1886‐343.1892‐240.1016‐104.1089

17 C47H80O17 46.81 M −H½ �− KXS, GR 915.5323 915.5270 277.2151

18 C48H82O18 35.4 M+Na½ �+ KXS, GR 969.5393 969.5383 789.4727‐349.1107‐203.0524

19 C48H82O18 42.49 M+Na½ �+ KXS, GR 969.5393 969.5374 855.5226‐539.2863‐447.2406‐429.2286

20 C53H90O22 41.09 M+Na½ �+ KXS, GR 1101.5816 1101.5823 —

21 C54H92O23 40.74 M −H½ �− KXS 1107.5956 1107.5956 945.5487‐783.4916‐325.1803‐179.0564

22 C54H92O23 40.95 M+Na½ �+ KXS, GR 1131.5922 1131.5929 789.4755‐365.1054

23 C54H92O23 45.99 M+H½ �+ KXS 1109.6102 1109.6129 —

10 Oxidative Medicine and Cellular Longevity

time, it also conforms to the pathological mechanisms ofmost diseases caused by multiple causes, especially chronicdiseases and complex diseases [38]. The selection of com-

pound formulae is based on the integral theory of TCMand follows the rule of drug compatibility and synergism[39]. However, it is still a great challenge to find out the directtargets and explore the mechanisms of multitarget and mul-tichannel actions of the TCM compound. Trying to explorethe possible working mechanism of the formula with systemsbiology approaches, we used KXS as an example since it iscertified to have a curative effect in treating AD.

In our study, it was found that, in the AD rat modeltreated with KXS consisting of four single herbs, there wasa significantly shortened escape latency and swimming dis-tance in the Morris water maze to improve cognitive

Table 2: Identification of the components of Kai-Xin-San in brain samples of rats.

No. Formula tR (min) Ion addition Source Cal m/z m/z Fragment ions (m/z)

1 C12H16O4 20.60 M+H½ �+ KXS 225.1121 225.1149 —

2 C12H16O4 28.18 M+Na½ �+ KXS 247.0991 247.1049202.0811‐172.9562‐131.0022‐116.9332‐

88.0237‐80.0488‐56.9698

3 C12H16O5 20.27 M+Na½ �+ KXS 263.0890 263.0805 78.9849‐60.9592‐56.9690

4 C12H16O5 40.76 M+H½ �+ KXS, GR 241.0971 241.0931 153.0531‐91.0597

5 C16H20O9 37.93 M −H½ �− KXS 355.1035 355.1012 297.1512‐183.0114‐119.0531

6 C21H24O11 9.75 M+Na½ �+ KXS 475.1211 475.1203 —

7 C21H24O11 40.30 M+H½ �+ KXS, GR 453.1391 453.1391 328.2223‐187.0355‐160.9726‐124.9905‐76.9988

8 C36H46O7 28.07 M+Na½ �+ KXS, GR 613.3136 613.3144 —

9 C36H46O7 45.93 M+H½ �+ KXS, GR 591.3316 591.3344 532.2582‐408.2573‐386.2741‐146.9808‐86.0980

10 C42H72O13 40.26 M+Na½ �+ KXS 807.4865 807.4872 —

Table 3: The correlation of pharmacological indicators and plasma components.

Source FormulaPearson’s correlation coefficient (r)

AChE ROS IL-1β TNF-α p-Tau (S199) p-Tau (S396) Crossing number

GR C16H20O9 -0.767 -0.703 -0.855 -0.723 -0.8 -0.744 0.675

GR C36H62O10 -0.751 -0.679 -0.846 -0.696 -0.783 -0.735 0.644

GR C47H80O17 -0.785 -0.731 -0.864 -0.756 -0.819 -0.754 0.714

GR C48H82O18 -0.832 -0.822 -0.867 -0.865 -0.867 -0.765 0.848

GR C48H82O18 -0.835 -0.85 -0.844 -0.903 -0.87 -0.75 0.9

GR C53H90O22 -0.762 -0.695 -0.852 -0.714 0.795 -0.741 0.665

GR C54H92O23 -0.566 -0.439 -0.711 -0.426 -0.591 -0.605 0.346

GR C41H70O14 -0.695 -0.602 -0.81 -0.607 -0.725 -0.699 0.544

PR C12H16O4 -0.823 -0.9 -0.698 -0.893 -0.782 -0.738 0.821

PO C12H16O5 -0.335 -0.412 -0.226 -0.519 -0.49 -0.299 0.524

PO C21H24O11 -0.025 -0.077 0.087 -0.182 -0.222 -0.052 0.164

PO C36H46O7 -0.03 -0.083 0.081 -0.188 -0.227 -0.056 0.171

PO C42H72O14 -0.598 -0.687 -0.506 -0.781 -0.687 -0.503 0.808

AT C10H12O5 0.04 -0.119 0.036 -0.21 0.137 0.285 0.509

AT C12H14O5 -0.152 -0.312 -0.137 -0.409 -0.075 0.093 0.678

AT C12H16O4 0.507 0.388 0.459 0.335 0.632 0.694 -0.033

AT C12H16O5 -0.032 -0.193 -0.029 -0.287 0.057 0.214 0.576

AT C22H30O14 -0.299 -0.454 -0.271 -0.553 -0.242 -0.064 0.787

AT C36H56O13 0.166 0.012 0.15 -0.072 0.274 0.404 0.382

Table 4: The number of components in four single herbs in ADrat plasma.

Number of cases GR PR PO AT

All 8 1 4 6

rj j > 0:5 7 1 1 0

11Oxidative Medicine and Cellular Longevity

impairment. In the KXS treatment group, AChE, a character-istic hydrolase of ACh, was significantly reduced and couldfurther regulate the level of ACh. These results, together withthe immunofluorescence of NeuN, suggested that KXS play acrucial role in the anti-AD therapeutic effect. Satisfactorily,KXS did not reveal any obvious side effect in AD rats, asshown by weight change, consistent with the meeting of theclinical medication safety committee. In order to explorethe possible working mechanisms, a study of the related pro-tein signaling pathways was particularly necessary [40].Plenty of researches have confirmed that Tau was the vitalpathogenic factor and important target for drug development[41, 42]. Interestingly, KXS exerted the best pharmaceuticaleffect in regulating Tau signaling pathways, and among thefour single herbs of KXS, GR has also been regarded as theprimary single herb aiming at Tau hyperphosphorylation.In AD rat research, KXS was discovered to be able to upreg-ulate p-PI3K, p-Akt, and p-GSK-3β and further lead to thedownregulation of p-Tau (S199 and S396) in the hippocam-pus of AD rats. In the cellular level, it was found that KXScould modulate Tau hyperphosphorylation by the PI3K/Aktsignal pathway, while Tau, as the microtubule-associatedprotein, could play a role on the cytoskeleton of nerve cellsthrough maintaining the stability of microtubules. Thetherapeutic effect of GR was the most effective among thefour single herbs, and the effects of GR coupled with PR,PO, or AT were further enhanced to a certain degree. Fur-thermore, Tau was not only regulated by phosphorylationbut also by truncation, glycosylation, acetylation, and otherposttranslational modifications [43]. In the pathologicalprocess of AD, the Tau protein could be truncated andcould induce the apoptosis of neurons. The enzymeinvolved in abnormal Tau truncation was mainly caspase-3, which also indicated that KXS might be related to theregulation of apoptosis [44, 45].

Maintenance of the normal physiological status requireda balance of the production and cleanup of free radicals [46].KXS could regulate oxidative stress both in vivo and in vitro;specifically, intracellular ROS could be reduced by GR, PR,PO, and AT, particularly when the four herbs were adoptedin combination. Furthermore, it was known that, in the syn-ergistic and compatible rule of designing the formula inTCM, some herbs should play an adjuvant role in facilitatingthe delivery of the sovereign drug to the disease site in vivo[47]. Then, formula dismantling in vitro indicated thatginseng was the principal herb of the formula, whereasthe other three herbs served adjuvant roles to achieve thebest antioxidant effect of the whole formula. Meanwhile,neuroinflammation was also an important inducement ofpathological changes in AD and interacted with oxidativestress [48, 49]. In the KXS treatment group, TNF-α andIL-1β, the critical proinflammatory factors, were signifi-cantly downregulated both in vivo and in vitro. It was foundthat GR showed the strongest antineuroinflammation effectin four single herbs and the effects of GR coupled with PR,PO, or AT were further enhanced to a certain degree. Theseresults suggested that PR, PO, and AT might assist GR inthe KXS formula through mediating neuroinflammation.Several studies suggested that a vicious circle between

oxidative stress and neuroinflammation was a significantpathogenic feature in AD, and the circle might aggravatethe Aβ-induced apoptosis and the hyperphosphorylation ofTau [50, 51]. In the AD model group, apoptosis-relatedproteins had high expression, whereas the KXS formula sig-nificantly inhibited the expressions of Bax, bcl-2, andcleaved-caspase-3. This phenomenon not only suggested thatKXS might regulate oxidative stress and neuroinflammation-triggered apoptosis but also indicated that the formulaemight have therapeutic effects through mechanisms beyondeach herb.

The application of the KXS formula has been retro-spected to the Tang dynasty, and the explanation of the basisof its potential pharmacodynamic material could also beachieved by modern analytical techniques [52]. At the smallmolecule level, the LC-Q-TOF-MS method with high resolu-tion and sensitivity was established to analyze the transitionalcomponents of KXS in vivo. The mass spectra analysisshowed that 23 components were firstly detected in AD rats’plasma, including 8 from GR, 1 from PR, 4 from PO, 6 fromAT, and 4 that only existed in KXS; meanwhile, 10 constitu-ents were firstly found in AD rats’ brain, including 4 fromGRand 6 that were only discovered in KXS. Through semiquan-titative analysis, it is suggested that the peak areas of somecomponents in the KXS group were larger than that in theGR group. Moreover, it was found that the herbs’ combina-tion could facilitate the transportation of GR into bloodand through the blood-brain barrier. The results not onlyverified the principal status of ginseng but also indicated thatthe formula might have better anti-AD effects than each herbin the formula. These phenomena reflected the synergisticeffect and the formula compatibility rationality of KXS tosome degree. In order to further explore the principles of for-mulating prescription, assessment of the coefficient value bythe Pearson correlation analysis was applied to explain thecorrelation between plasma components and pharmacologi-cal function. The correlation analysis results (Tables 3 and4) suggested that the components in GR showed the stron-gest correlation with anti-AD effects based on the values ofthe correlation coefficient (jrj > 0:5), as well as reflected theaction of the multicomponent, multichannel, and multitargetcharacteristics of traditional Chinese medicine.

In conclusion, the data not only suggested that KXS couldameliorate AD by regulating the cholinergic system, Tauhyperphosphorylation, oxidative stress, neuroinflammation,and apoptosis in the AD model but also indicated that GRis the principal herb of the formula, whereas PR, PO, andAT acted as adjuvant herbs. The results acquired by systemsbiology methods at the animal, cellular, and molecular levelssuggested that the dissection of the mechanism of the clini-cally well-used formula could provide a novel way in explor-ing the value of TCM.

Data Availability

The data used to support the findings of this study areincluded within the article and the supplementary informa-tion files.

12 Oxidative Medicine and Cellular Longevity

Conflicts of Interest

The authors announce no conflict of interest to the currentresearch.

Authors’ Contributions

Sirui Guo and JiahongWang contributed equally to the work.

Acknowledgments

This study was supported by the National Natural ScienceFoundation (No. 81473324). This study was also supportedby the Liaoning Innovative Research Team in University(No. LT2013022) and the Liaoning Distinguished ProfessorProject for Qing Li (2017) and the Shenyang PharmaceuticalUniversity Innovative Research Team.

Supplementary Materials

HPLC-QTOF MS was applied to establish a method forsemiquantitative analysis of components into plasma andbrain of AD rats. Data processing methods, such as themultiple product ion filtering technique and the neutral lossfiltering technique, were adopted, and the components wereassigned by formula disassembly. The peak areas of compo-nents after oral administration of KXS are listed in Table S1and Table S2. (Supplementary Materials)

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